1. Field of the Invention
Polymeric enzyme products, intermediates therefor, and their production.
2. Description of the Prior Art
Recent developments in the well established field of enzymes have been directed toward the development of stable, insoluble forms of enzymes which retain the activity of the natural enzyme. Several review articles have been published which summarize the preparation and applications of insolubilized enzymes: Orth et al., Angew. Chem. 11, 249-346 (1972); Mosbach, Acta Chem. Scand. 24, 2084-2092 (1970); Mosbach, Sc. Amer. 224, 26-33 (1971); Kay, Proc. Biochem. 3, 36-39 (1968); Goldstein et al., Z. Anal. Chem. 243, 375-396 (1968); Goldstein, "Methods in Enzymology," Academic Press, N.Y. (1971); Vol. 19, pp. 935 ff; Silman et al., Ann. Rev. Biochem., 35, 873-908 (1966); Katchalski et al., Advan. Enzymol. 34, 445 (1971). A rather comprehensive summary of the state of the art is presented in U.S. Pat. No. 3,650,900, issued Mar. 21, 1972, and by Melrose, Rev. Pure and Appl. Chem., 21, 83-119 (1971).
The various methods employed for the insolubilization of enzymes by attachment to or on a matrix fall into four principal methods: (a) covalent chemical linkage via functional groups of the enzyme that are not essential to enzyme activity; (b) entrapment or inclusion of the enzyme within a hydrophilic gel lattice which retains the enzyme but allows substrate and product to pass through; (c) ionic binding (physical adsorption) on hydrophilic ion exchangers, or on charcoal or glass beads; and (d) cross-linking them into large aggregates by reaction with bifunctional compounds.
Insoluble (immobilized) enzymes and their preparation are described in the following patents:
______________________________________ U.S. 3,536,587 U.S. 3,650,900 U.S. 3,574,062 U.S. 3,650,901 U.S. 3,607,653 British 1,224,947 U.S. 3,616,229 British 1,274,869 U.S. 3,619,371 German 1,935,711 U.S. 3,645,852 German 2,012,089 ______________________________________
Ionic binding is not a reliable technique when a totally insoluble preparation is desired since partial or total desorption of enzyme may result from a change in ionic strength, pH or temperature, or addition of substrate.
The insolubilization of enzymes by inclusion techniques is not completely satisfactory since small amounts of enzymes leak out from such prepared gels.
The covalent linkage of enzymes to insoluble carriers offers a method of preparing water-insoluble derivatives which will not be solubilized when used or when the composition of the medium is changed provided that the covalent linkages formed are such as will not be broken under the conditions of biochemical use.
In general, the binding of a biologically active protein to an insoluble carrier by covalent bonds must be carried out via functional groups on the enzyme which are non-essential for its biological activity. The binding reaction should obviously be performed under conditions which do not cause denaturation.
Various physical attributes of the carrier such as solubility, mechanical stability, swelling characteristics and porosity, as well as its electric charge and hydrophilic or hydrophobic nature, play a major role in determining the maximal amount of enzyme which can be covalently bound, and the stability and biological activity of the insoluble product. Minimal solubility, high mechanical stability and adequate particle size are essential for the preparation of biologically-active, bound enzymes which can be readily and completely removed from reaction mixtures by filtration or centrifugation. Similar requirements must be met in the preparation of stable, biologically-active columns with well defined flow rates.
Reported immobilized enzymes include, for instance, hydrolytic enzymes such as trypsin, chymotrypsin, pepsin, pancreatin, papain, fungal and bacterial proteases, amino acid acylase, ribonuclease, phosphatase, pectinase, invertase and amylase. Of special interest is the immobilization of penicillin acylase and glucose oxidase, the enzymes responsible for the hydrolysis of a penicillin to 6-aminopenicillanic acid and a carboxylic acid, and for the conversion of glucose to glucono delta lactone, respectively. The 6-aminopenicillanic acid is used in the synthesis of "new penicillins." The chemical attachment of penicillin acylase to derivatives of cellulose and the kinetics of these immobilized preparations are described in Biotechnology and Bioengineering 11, 337-348 (1969). Other immobilized preparations of penicillin acylase are covered in British patent specification Nos. 1,183,257; 1,183,258; 1,183,260; and 1,193,918. The kinetic behavior and covalent linkage of glucose oxidase to porous glass is described in Biochemical Journal 124, 801 (1971). Glucose oxidase chemically attached to insoluble siliceous particles is discribed in U.S. Pat. No. 3,519,538.
The object of this invention is to provide new, stable, immobilized enzyme preparations with high enzyme incorporation and high enzyme activity. These stable preparations can be used repeatedly without significant loss of enzyme activity.